Field of the Invention
The invention in the field of biochemistry, molecular biology and medicine relates to the suppression of growth and metastasis of malignant pulmonary mesothelioma (MPM) cells and the treatment of MPM using nucleic acid vectors, or endothelial protein C receptor (EPCR) agonists in the form of polypeptides, peptides or small organic molecules.
Description of the Background Art
It has been well recognized for many decades that tumors dramatically increase the risk for hemostatic abnormalities such as disseminated intravascular coagulation, pulmonary and venous thromboembolism (1-3). Prior studies have also established that hemostatic factors play a major role in cancer biology, particularly in tumor dissemination and metastasis (4-6). Tumor cell-associated tissue factor (TF) is known to contribute to tumor growth and progression either directly by TF-FVIIa or TF cytoplasmic tail-mediated cell signaling (7-9) or indirectly through generation of thrombin that leads to activation of platelets, fibrin deposition and activation of protease-activated receptor-1 (“PAR1”)-mediated cell signaling (10-13). Consistent with the importance of thrombin in tumor biology, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM), two transmembrane glycoproteins present on endothelial cells that impair thrombin generation were found to diminish tumor metastasis (14-16).
EPCR belongs to the Class 1 MHC family of receptors. TF belongs to Class 2 cytokine receptor family. Both have short cytoplasmic tails implicated in cell signaling. EPCR is constitutively expressed in endothelium, acts as the receptor for anti-coagulant protein C cofactor to enhance cleavage of clotting factors Factor V (FV) and Factor VIII (FVIII). EPCR was subsequently found to be expressed on many cell types. TF is constitutively expressed on perivascular cells and cancer cells. TF is a cofactor for FVIIa that initiates the clotting cascade. In response to inflammatory stimuli, TF is induced in many cell types. The role(s) of EPCR and TF on tumor growth in general, and of malignant pleural mesothelioma (MPM) in particular, are largely unknown.
Mesothelioma is a rare but serious cancer of the membranous tissue that covers majority of internal organs. Most often it occurs in the pleural membranes covering lungs, heart and diaphragm and is called the pleural mesothelioma. It is also found in the peritoneum and testicular membrane. Mesothelioma is prevalent in workers and handlers of asbestos. Most mesothelioma patients die within 4-18 months after diagnosis. Five year survival is only 5-10% and so far there is no cure.
Recent studies showed promotion of tumor growth by TF independent of its role in coagulation (17-20). Selective inhibition of TF-FVIIa signaling using a specific monoclonal antibody (“mAb”)—that blocks TF signaling but not TF-mediated coagulation was shown to reduce breast tumor growth (17). Blockade of protease activated receptor-2 (“PAR2”) cleavage but not PAR1 cleavage by specific antibodies attenuated tumor growth. Consistent with the hypothesis that PAR2-mediated signaling contributes to tumor growth in breast cancer, mice lacking PAR2 but not PAR1, exhibited reduced tumor growth in a model of spontaneous mammary tumors (21). However, it is presently unknown whether TF-FVIIa-PAR2 signaling is responsible for TF-driven tumor growth in other types of cancers. The present inventors' recent studies on progression of MPM in nude mice showed that MPM cells that express TF generated large tumors within the pleural cavity and inhibition of tumor cell TF by overexpression of TFPI by tumor cells blocked tumor growth and invasion (22). It is presently unknown whether TF-FVIIa-PAR2-mediated cell signaling contributes to growth of MPM as was observed in breast cancer (17, 21).
The present inventors' laboratory and others have recently established that FVIIa, the clotting factor that initiates the activation of the coagulation cascade upon binding to TF, also binds to EPCR (23-25). FVIIa binding to EPCR on the endothelium or in cells expressing TF induced cell signaling by activating PAR1 either directly (26) or by enhancing TF-FVIIa-FXa cleavage of PAR1 (27). Studies in cell model systems implicated EPCR in tumor metastasis. PAR1 signaling mediated by interaction between EPCR and activated protein C (“APC”) was shown to promote cancer cell migration, invasion and angiogenesis (28, 29). In vivo studies gave conflicting results as EPCR-APC signaling decreased lung metastasis in a melanoma model system by preventing tumor cell migration through enhancement of endothelial barrier function (15, 30) whereas EPCR overexpression increased metastasis in lung adenocarcinoma by promoting tumor cell survival (31). Prior to the present invention, there was no information on whether EPCR directly influences tumor growth.
TF has been found recently to contribute actively to tumor growth through a nonhemostatic, TF-dependent signaling mechanism in melanoma (19) and breast cancer (17, 21). TF supported tumor growth in breast cancer via TF-FVIIa-PAR2-mediated cell signaling, independent of PAR1 (17, 21). However, it was unclear whether such a mechanism is also responsible for tumor growth in other types of cancers. TF, in addition to facilitating TF-FVIIa binary complex-mediated activation of PAR2, can also support TF-FVIIa-FXa ternary complex activation of PAR1 (36). Moreover, studies from the inventors' laboratory and others showed that TF-FVIIa binary complex can also activate PAR1 (37).
The present inventors know of no prior reports concerning the influence of EPCR on tumor growth, though a number of studies reported that EPCR-APC signaling exerts anti-apoptotic effects on a variety of cell types (45-50). The APC/EPCR axis conferred a significant advantage in cell survival to lung adenocarcinoma cells, and this was responsible for robust prometastatic activity (31). Based upon such observations, one might have concluded that EPCR, just as TF, promotes tumor growth. However, as disclosed herein, EPCR, in fact, suppresses, tumor growth. This unexpected and novel finding runs contrary to the known functions of EPCR.